The cytochrome P450 hemoproteins catalyze the oxidation of many chemicals, including xenobiotics, drugs, and endogenous substrates. The P450s are of considerable toxicological and therapeutic interest due to their broad and overlapping substrate specificity, and although progress has been made in the elucidation of the individual steps of P450 catalysis, it is still unclear what factors determine reaction pathways and rates of product formation. The goal of the proposed work is to determine the rate-limiting step(s) in P450 2A6 catalysis using the carcinogen N,N-diethylnitrosamine (DEN) as a substrate. DEN is oxidatively converted by P450s 2A6 and 2E1 to a reactive DNA-alkylating diazonium ion, and studies by others have shown that deuterium-substituted dimethylnitrosamine is a less potent carcinogen than the fully protiated form. Previous work done in this laboratory indicates a rate-limiting step between product formation and release in P450 2E1-catalyzed ethanol oxidation, but it is not clear if this paradigm holds for other P450 enzymes. A model of the P450 2A6 catalytic cycle will be constructed that is consistent with kinetic isotope effects (steady-state and pre-steady-state), "burst" rates of product formation, and rates of intermediate reactions in the catalytic oxidation of DEN. Limits of the model will be examined by computer simulation and fitting of experimental data.